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Mikhail A. Nikiforov (ed.), Oncogene-Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 1534, DOI 10.1007/978-1-4939-6670-7_5, © Springer Science+Business Media New York 2017
Chapter 5
Genome-Wide miRNA Screening for Genes Bypassing Oncogene-Induced Senescence
Maria V. Guijarro and Amancio Carnero
Abstract
MicroRNAs are small noncoding RNAs that regulate gene expression by binding to sequences within the 3′-UTR of mRNAs. Genome-wide screens have proven powerful in associating genes with certain phenotypes or signal transduction pathways and thus are valuable tools to defi ne gene function. Here we describe a genome-wide miRNA screening strategy to identify miRNAs that are required to bypass oncogene- induced senescence.
Key words miRNA , Screening , Cellular senescence , Oncogene-induced senescence
1 Introduction
Cellular senescence is a state of irreversible proliferative quiescence characterized by changes in cytoplasmic and nuclear morphology, DNA-damage signaling, transcription, chromatin conformation, and a strong increase in the secretion of pro-infl ammatory cyto- kines [ 1 – 3 ]. Senescence is the fi rst line of defense against poten- tially transformed cells, and the progression to malignancy correlates with a bypass of cellular senescence termed “immortal- ization” [ 4 – 7 ]. Senescence has been observed in vitro and in vivo in response to various stimuli, including oncogenic stress, oxidative stress, and chemotherapeutic agents [ 8 – 12 ]. Cells with cellular and molecular characteristics of senescence—terminally arrested at G1 with increased levels of many cell cycle inhibitors—have been found to be associated with the activation of oncogenes and the inactivation of tumor suppressor genes in precancerous lesions [ 13 – 17 ]. The inactivation of senescence effectors in parallel to oncogenic activation results in cancerous growth progression [ 14 , 18 – 20 ]. Senescence activation can be considered to be a cellular response to cell damage [ 5 ]. The pathways involved in cellular senescence exhibit several levels of regulation with redundancy 1.1 Cellular
Senescence
among them. Moreover, signal transduction through canonical signaling pathways and additional layers of regulation by miRNAs and methylation have been recently discovered [ 21 , 22 ]. The shortening of telomeres has been proposed to be the “clock”
responsible for counting divisions in human cells and limits the number of duplications [ 23 ]. In general, most tumors contain telomeres elongated by telomerase activity, which allows the con- stitutive elongation of telomeres. Telomerase activity is essential for replicative immortality in humans but not in most murine models [ 9 ]. Cellular senescence can also be elicited by other types of stress, including oncogenic, redox, and DNA-damage stresses, but in these cases, the establishment of cellular senescence is also independent on telomerase [ 24 ].
Morphologically, senescent cells show as fl at and enlarged and are commonly multinucleated [ 25 ]. They present nuclear envelope alterations in senescence nuclear structures, such as the nuclear lamina, nucleoli, the nuclear matrix, and nuclear bodies (such as promyelocytic leukemia bodies). It is especially interesting that multinucleation is likely a consequence of the failure of nuclear envelope breakdown. Lysosome / vacuoles also show alterations by a decrease in the lysosomal recycling capacity for proteins, lipids, and mitochondria [ 26 ]. Consequently, accumulation of damaged mitochondria lowers ATP production and elevates ROS produc- tion. It is thought that the senescence-associated β-galactosidase (SA-β-gal) activity, which is detected by histochemical staining of cells with the artifi cial substrate X-gal, is due to the altered lysosomal content [ 27 , 28 ].
Senescent cells also display molecular features of DNA dam- age. Markers of DNA-damage response localize at telomeres in senescent cells after serial passage, which indicates that the DNA- damage response can be triggered by telomere shortening [ 29 – 31 ]. These markers include nuclear foci of phosphorylated histone H2AX, and the localization at double-strand break sites of DNA repair and DNA-damage checkpoint factors, such as 53BP1, MDC1, and NBS1 [ 29 , 32 ]. Senescent cells also contain activated forms of the DNA-damage checkpoint kinases Chk1 and Chk2.
These characteristics also explain why other DNA-damage stress- ors, such as culture shock, potentially initiate senescence without telomere involvement [ 33 ].
The genomic methylation status generally declines during cellular senescence. Hypomethylation has been observed in both replicative and premature senescence, suggesting that genome hypomethylation is necessary to confer an unstable internal envi- ronment and conceivably promotes cellular senescence [ 21 , 34 ]. In this regard, the initiation of senescence triggers the generation and accumulation of distinct heterochromatic structures, known as senescence-associated heterochromatic foci (SAHF) . The formation of SAHF coincides with the recruitment of heterochromatic
proteins and the pRb tumor suppressor to E2F-responsive promoters.
SAHF accumulation is associated with stable repression of E2F target genes and does not occur in reversibly arrested cells. SAHF formation and promoter repression depend on the integrity of the pRb pathway [ 35 ]. These results provide an explanation for the stability of the senescent state. Accordingly, with these results, genome-wide expression analysis indicates that genes whose expression is upregulated during replicative senescence in human cells are physically clustered [ 36 , 37 ].
Large protein and lipid modifi cation is another characteristic of senescent cells [ 38 , 39 ]. Oxidation, glycation, cross-linking, and other chemical modifi cations impair the molecular functions of multiple vital components, including DNA, membranes, the extra- cellular matrix (ECM), enzymes, and structural proteins.
Modifi cations that accumulate faster than they are repaired or recy- cled will cause progressive deterioration over time.
Another main characteristic of senescent cells is the senescence- associated secretory phenotype (SASP) . Senescent cells undergo widespread changes in protein expression and secretion, which ulti- mately derive into the SASP [ 40 , 41 ]. Senescent cells upregulate the expression and secretion of several matrix metalloproteinases that comprise a conserved genomic cluster and interleukins that promote the growth of premalignant epithelial cells. A limited number of cell culture and mouse xenograft studies support the idea that senescent cells secrete factors that can disrupt tissue structure, alter tissue function , and promote cancer progression [ 42 – 44 ].
As mentioned, in addition to telomere dysfunction , cellular senescence can be elicited by other types of stress, including onco- gene activation [ 45 ]. This phenomenon is not observed for onco- genic RAS exclusively; many, but not all, of its effectors, including activated mutants of RAF, MEK, and BRAF , were shown to cause senescence as well [ 46 – 49 ]. Some oncogenes, such as RAS, CDC6, cyclin E , and STAT5, trigger a DNA-damage response (DDR) , associated with DNA hyper-replication that appears to be causally involved in oncogene-induced senescence (OIS) in vitro [ 50 – 53 ].
During the last decade, OIS has been studied predominantly in cell culture systems, triggering a long debate as to whether or not OIS corresponds to a physiologically relevant phenomenon in vivo. In favor of OIS representing an in vitro phenomenon only is that artifi cial conditions, such as the use of bovine serum and plastic dishes, as well as the presence of supraphysiologic O 2 , generate a stress signal that at the very least contributes to triggering a cellular senescence response [ 54 , 55 ]. However, conversely, senescence bypass screens have identifi ed several genuine human oncogenes, including TBX2, BCL6, KLF4, hDRIL, BRF1, PPP1CA, and others [ 56 ]. Furthermore, virtually all human cancers lack func- tional p53/pRB pathways and two key senescence-signaling routes [ 57 ] and often carry mutations in sets of genes known to collabo- rate in vitro in bypassing the senescence response.
The dynamics of senescence exhibit two different steps: cell cycle arrest and further acquisition of senescence features, which include permanent arrest, termed geroconversion [ 1 , 58 ].
Senescence effector pathways converge at the point of cell cycle arrest through cyclin-dependent kinase (CDK) inhibition.
Therefore, most pathways known to be involved in senescent arrest impinge either directly or indirectly on this process. Namely, the most known effector pathways are the p16 INK4a /pRB, the p19 AR F /p53/p21 CIP1 , and the PI3K/ mTOR /FoxO pathways, all of which exhibit a high degree of interconnection [ 5 , 56 , 59 – 61 ].
However, two pathways have been proposed to be responsible for the acquisition of irreversible arrest and geroconversion: the pRB and the mTOR pathways. If geroconversion is not activated, cells are only transiently arrested with the possibility of resuming growth once the proliferation constraints have been eliminated [ 32 , 62 ]. It has also been shown that if mTOR is activated under conditions of proliferative arrest, the arrest becomes permanent and the cell undergoes senescence [ 63 , 64 ]. This can also be accomplished by producing permanent changes in the chromatin, especially at E2F transcription sites, which result in a blockade of transcription of proliferative genes [ 35 ]. It has been shown that permanent inactivation of pRb, perhaps in combination with phosphatases, may signal for the differential recruitment of silenc- ers to the heterochromatin of promoter sites [ 65 ]. Human cells show heterochromatin compaction during senescence, SAHF , which is dependent on the pRb pathway. These SAHFs cause stable silencing of cell cycle genes and seem to be a factor in the stability of permanent arrest during senescence [ 66 ]. Also, the role of senescence in embryonic development seems to be depen- dent on the pRb pathway through CDK inhibitors p21 CIP1 and p15 INK4b but independent of other cell cycle inhibitors, DNA damage , or p53 . This senescence during embryonic development seems also regulated by the PI3K/FOXO and TGFb/SMAD pathways [ 67 , 68 ].
It is therefore clear that epigenetic alteration (e.g., by means of miRNAs) of any of the effector pathways may have an effect on the senescence onset contributing to cellular immortalization .
MiRNAs are a class of noncoding RNAs that can act as potent oncogenes and tumor suppressors, playing crucial roles in the initiation, maintenance, and progression of the oncogenic state in a variety of cancers [ 69 ]. These small (~18 to 25 nucleotides) RNAs can bind target mRNAs in a sequence-specifi c fashion to induce their posttranscriptional downregulation. This binding is dependent on the “seed sequence,” a 6–8 nucleotide sequence at the 5′ end of each miRNA that is complementary to sites found in the 3′-untranslated region (UTR) of target mRNAs (target sites).
It is thought that individual miRNAs can target multiple genes for 1.2 Effector
Pathways
1.3 miRNAs
regulation and that these targets are extremely diverse in cellular function, suggesting that miRNAs play important roles in a wide variety of cellular processes [ 70 – 73 ].
From a functional genomics perspective, the often-convergent action of miRNAs and the fact that the relatively low number of miRNAs modulates a large fraction of the transcriptome provide a unique opportunity to interrogate the genome through the devel- opment of phenotypic gain- or loss-of-function miRNA screen- ings and thus gain mechanistic insights into complex biological and disease-relevant processes. It also provides the opportunity to identify genes involved in phenotypes that arise from the simulta- neous targeting of multiple genes; these are not accessible through classic genetic or RNA interference (RNAi) screening methods that rely on the phenotypic analysis of single mutations or knockdowns .
Despite the reported discrepancies between the upregulation and downregulation of miRNAs during aging and cellular senes- cence, such as miR-34a, several miRNAs are differentially expressed in senescent cells when compared to primary cells, which implies a role for miRNAs in senescence [ 74 , 75 ]. Some miRNAs (including miR-20a, miR-24, miR-34a, miR-106a, and miR-449a) that funnel proliferating cells to senescence regulate cellular senescence via either or both p53/p21 and p16/pRb pathways [ 76 ]. The coordi- nated action between senescence-associated miRNAs in p53/p21 and p16/pRb pathway with transcription factors (Myc and E2F) in cell cycle regulation contributes to the inhibition of cell prolifera- tion during cellular senescence [ 77 ]. The miRNAs control cell tran- sition, mainly through the G1/S checkpoint during cell cycle progression by targeting the components of cell cycle including cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CDKIs) [ 78 ].
miR-29 and miR-30 directly repress B-Myb in conjunction with Rb-E2F complexes, to induce senescence [ 79 ]. miR-29 has also been reported downregulated in cell lymphomas [ 80 ], and its over- expression is suppressed during tumorigenicity in lung cancer cells [ 81 ]. Recently, miR-34a overexpression has been shown to induce senescence in a p53 -independent manner through repression of c-Myc [ 82 ]. However, it is found downregulated in neuroblastomas, pancreatic, colon, and lung cancer cells, suggesting its involvement in cellular immortalization [ 83 , 84 ].
miR-449a suppresses pRb phosphorylation inducing senes- cence [ 85 – 87 ]. In a recent study, miR-449a is shown to be down- regulated in prostate cancer, regulating cell growth and viability, in part by repressing the expression of HDAC-1 [ 87 ]. miR-128a directly targets the Bmi-1 oncogene (polycomb ring fi nger onco- gene; BMI1), which increases p16 INK4A expression and reactive oxygen species (ROS) . Collectively, these effects promote cellular senescence in medulloblastoma cell lines. miR-217, expressed in
endothelial cells during aging, promotes premature senescence by inhibiting SIRT1 expression. This occurrence increases Forkhead box O1 (OXO1) expression [ 88 ]. In addition, miR-217 has been reported to be a novel tumor-suppressive miRNA that targets K-Ras in pancreatic ductal adenocarcinoma due to decreases in tumor cell growth both in vitro and in vivo [ 89 ]. miR-20a induces senescence in MEFs by directly downregulating the transcriptional regulator leukemia/lymphoma-related factor (LRF), which induces p19 ARF [ 90 ]. In addition, miR-519 induces senescence in cancer cell lines by repressing HuR expression [ 91 ].
In contrast, there are miRNAs that are downregulated during senescence, such as miR-15b, miR-24, miR-25, and miR-141, which directly target mitogen-activated protein kinase kinase (MKK4) [ 92 ]. Recently, it was shown that 28 miRNAs prevented senescence induced by oncogenic Ras G12V [ 90 ]. These miRNAs bypass Ras G12V -induced senescence by directly targeting the 3′-UTR of p21 Cip1 . Moreover, miR-372, miR-373, miR-302, and miR-520 also bypass Ras G12V -induced senescence through the downregulation of LATS2 in addition to p21 Cip1 [ 90 ]. These identifi ed proliferative miRNAs are associated with cancer development [ 22 , 93 ].
Modulation of intracellular miRNA levels can be achieved through transfection of synthetic miRNA hairpin precursors or duplex miRNA mimics (to increase miRNA levels), or of oligonucleotides, designated miRNA inhibitors that are designed to sequester mature miRNA sequences and thus decrease the availability of a particular miRNA. Different genome-wide libraries are available from various commercial sources (Table 1 ). The design of miRNA mimics fre- quently involves chemical modifi cation of the passenger strand to minimize their incorporation in miRNA -induced silencing com- plex (miRISC) and thus better discriminate the phenotypic conse- quences of modulating each mature miRNA individually. Although the nature of these modifi cations is in most cases proprietary, this is an important point that should be considered. The use of miRNA hairpin precursors may be more representative of the physiological setting because these are processed by the cell machinery, but it renders analysis of the effect of each miRNA strand diffi cult to defi ne because potentially both miRNAs strands can be active.
The miRNA screen described below is based upon the study of Borgdorff et al. in which they identify miRNAs preventing Ras GV12 - induced senescence in human mammary epithelial cells (HMECs) [ 90 ]. In brief, they use a positive-selection strategy to identify miRNAs that when overexpressed, rescued the cells from Ras GV12 - induced senescence and demonstrate that this is achieved by pre- vention of Ras GV12 -induced upregulation of p21 Waf1/Cip1 . HMECs were infected with a retrovirus expressing 4-hydroxy-tamoxifen (OHT)-inducible oncogenic Ras (HMEC-ER-Ras GV12 ). After selection they were reverse transfected with the pre-miR miRNA library (Ambion), which consists of 328 miRNA mimics. Cells that 1.4 Screenings